Utilizing in vitro assays to assess drug-induced central nervous system liabilities

Abstract

Central nervous system (CNS) liabilities (e.g. seizures) remain a significant cause of drug development attrition, yet they are typically not identified until late-stage in vivo toxicology studies where adverse findings can result in costly delays or project termination. Further, ~25% of clinical trial failures are due to safety concerns that were undetected in earlier in vivo studies. This emphasises the urgent need for predictive, human-relevant in vitro assays that can be implemented earlier in the drug development process to assess CNS safety risks. The cardiovascular safety field has benefitted from early-stage screening using human ion channel assays and human induced pluripotent stem cell (hiPSC)-cardiomyocytes. Similarly, we developed two complementary in vitro CNS safety assays: 1) an ion channel panel evaluated via automated electrophysiology, and 2) an hiPSC-neuronal model analyzed using microelectrode array (MEA) to measure electrical activity in response to known seizurogenic compounds. In addition to seizure risk, sedation is a common adverse finding in preclinical studies. To address this, we aimed to validate our in vitro CNS assay for predicting sedative compound effects. GABAergic signalling plays a crucial role in the sleep-wake cycle, with GABA_A receptor activation resulting in sedation. We tested 14 sedative compounds (targeting both GABA and non-GABA pathways) and 5 non-sedative controls in the hiPSC-neuronal model. MEA analysis showed that GABA_A agonists (muscimol, etomidate, ethanol) and certain ion channel antagonists (valbenazine, dextromethorphan, primidone) reduced neural firing and burst activity, indicating sedative effects. Negative control compounds produced no significant changes. Of the 19 known compounds screened, 85% were accurately classified. Automated patch-clamp electrophysiology further confirmed these findings by testing compound interactions with GABA_A and other CNS ion channels. These results support the use of our in vitro CNS models for detecting sedative and seizurogenic effects, thus enhancing translational relevance. These platforms offer valuable human-based tools for early hazard identification and prioritisation of drug candidates, as well as interpreting CNS-related findings from in vivo studies thus complying with the principles of 3Rs to minimise and replace the use of animals in scientific testing. Ongoing work is focused on expanding this model to evaluate additional sedation-related ion channels.